The long-term goal of this grant is to establish a comprehensive understanding of post-transcriptional controls critical to mammalian erythroid differentiation. Erythroid differentiation is marked by a set of dramatic morphologic and functional changes, much of this process occurring in a transcriptionally-silent environment. For this reason erythroid differentiation is heavily dependent on post-transcriptional controls. RNA binding proteins (RBPs), the major regulators of post-transcriptional controls, impact on transcript processing in the nucleus as well as the stability and function of the mRNA in the cytoplasm. RBPs vary widely in their structures, binding specificities, and cellular compartmentalization and can modulate RNA functions by direct actions and/or via the recruitment of effector complexes. Identification of RBPs, their mRNA targets, and corresponding post-transcriptional controls have advanced our understanding of numerous developmental processes and have revealed unanticipated pathophysiologic pathways. By defining post-transcriptional controls in erythroid differentiation, we will expand our understanding of inherited and acquired disorders of erythropoiesis and establish a template for similar investigations in other systems. To achieve this goal, we will carry out a novel and state-of-the-art transcriptome-wide analyses to reveal the full range of mRNA-protein (mRNP) interactions that accompany the dynamic process of erythroid terminal differentiation. This approach combines biochemical methodologies with innovative informatic pipelines specifically designed for comprehensive descriptions of complex RNA-protein interactions. This unbiased analysis will be combined with a series of targeted in-depth mechanistic studies that focus on two sets of mRNA/protein interactions that we have identified to play central roles in erythroid differentiation. The combined output of these complementing approaches will establish the unique network of post-transcriptional controls in this developmentally robust and clinically relevant model system. This proposal encompasses three Specific Aims. Aim 1. Characterize the roles of the polyC-binding proteins, ?1 and ?2, as post- transcriptional integrators of erythroid differentiation. Aim 2. Identify the critical role(s) of the polyA binding protein, PABPC, in defining and sustaining the erythroid transcriptome. Aim 3. Map the global structure of mRNA/protein interactions in erythroid cells and define the dynamic nature of these interactions in the differentiation process. Combining transcriptome-wide analyses with targeted mechanistic studies will generate a powerful access to the complex array of RNA/protein interactions that constitute critical determinants of erythroid differentiation. Success in these studies will fundamentally alter our understanding of this intensively studied pathway; serve as a prototype for investigations of post- transcriptional controls in other systems, and present novel targets fo future diagnostic and therapeutic innovations.